Most bacterial species produce secreted proteins that function to exploit biological niches (e.g., host systems) or in the metabolism of a variety of environmental energy sources. The biotechnology industry has taken advantage of these organisms to produce secreted recombinant proteins of commercial value. An advantage of secreted proteins is that large quantities of recombinant proteins can be easily purified away from the bulk of the bacterial proteins that remain cell-associated. Gram-positive species, such as Bacillus subtilis, are particularly useful because of the absence of an outer cell membrane that can complicate the purification of secreted products.
A major limiting factor that the industry faces is the growth yield that can be achieved using these recombinant organisms. During aerobic, exponential growth in the presence of glucose, bacteria metabolize glucose via glycolysis and inhibit the TCA cycle. Consequently, the products of glycolysis are converted into acetate and secreted into the extracellular fluid for use once the glucose is depleted from the medium. This secreted acetate is converted into acetic acid, which becomes toxic at high levels, inducing cell death. Current industrial technology often relies on the removal of the secreted acetate during growth, thus allowing the bacteria to reach higher cell densities and produce increased protein yields. Unfortunately, the removal of acetate from the medium is costly and can outweigh the financial benefits of achieving higher growth yields. An alternative strategy involves achieving a careful balance between the amount of carbon source utilized and the amount of acetate secreted. However, the maximum growth potential that is possible with higher levels of glucose is, ultimately, negatively impacted by the increased cell death induced by toxic acetate byproducts that are generated by growth in the presence of this carbon source.
The cid and lrg operons of S. aureus affect extracellular murein hydrolase activity and penicillin tolerance (Brunskill and Bayles, J. Bacteriol. 178(3): 611-618, 1996; Groicher et al., J. Bacteriol. 182: 1794-1801, 2000; Rice et al., J. Bacteriol. 185: 2635-2643, 2003). Disruption of the lrg operon in the laboratory strain RN6390 increased extracellular murein hydrolase activity and decreased penicillin tolerance, whereas disruption of the cid operon decreased extracellular murein hydrolase activity and increased penicillin tolerance (Groicher et al., J. Bacteriol. 182: 1794-1801, 2000; Rice et al., J. Bacteriol. 185: 2635-2643, 2003).
The lrgA and cidA gene products display structural similarities to the bacteriophage holin family of proteins, which control the timing and onset of bacteriophage-induced cell lysis (Brunskill & Bayles, J. Bacteriol. 178(19): 5810-5812, 1996; Bayles, Trends Microbiol. 8(6): 274-278, 2000; Groicher et al., J. Bacteriol. 182: 1794-1801, 2000; Rice et al., J. Bacteriol. 185: 2635-2643, 2003). Based on these similarities, along with the phenotypic consequences of the cid and lrg mutations, it is likely that the lrgA and cidA gene products regulate murein hydrolase activity in a manner analogous to antiholin and holins (inhibitor and effector holins), respectively (Bayles, Trends Microbiol. 8(6): 274-278, 2000; Rice & Bayles, Mol Microbiol 50: 729-738, 2003).
In view of the relationship between acetic acid regulation and cell death, there exists a continuing need for methods, cells, and compositions that can be used to reduce acetic acid production in bacterial cell culture, thereby enabling growth to higher cell densities and increased protein yields. The present disclosure addresses this need and provides unexpected benefits with respect to controlling acetic acid metabolism and regulating cell death in bacteria.